Understanding the molecular and cellular behavior in the live tissue (ex vivo) and live animal (in vivo) environment represents a frontier of current biomedical science. Our long term goal is building novel nonlinear optical (NLO) microscopy tools to explore the working mechanisms of biological systems at the live tissue and live animal level and to investigate the underlying molecular pathways in diseases. This proposal focuses on a significant and challenging topic - the central nervous system (CNS) which includes brain and spinal cord. The specific aims are: 1) Development of a compact multimodal multiphoton microscope and noninvasive imaging of live CNS tissues. Using a multiphoton imaging system that consists of three Ti:sapphire lasers and two laser scanning microscopes, we have demonstrated coherent anti-Stokes Raman scattering (CARS) imaging of myelin sheath, sum frequency generation imaging of astroglial processes, and two-photon excitation fluorescence imaging of calcium ion distribution in live spinal tissues. We will construct a compact multimodal NLO microscope using spectral detection and optical parametric generation. 2) Development of in vivo multimodal multiphoton microscopy. We have demonstrated 3D in vivo imaging of myelin sheath and collagen fibrils in a mouse sciatic nerve. We will build a video rate scanner for high- resolution CARS imaging of spinal cord and brain in live animals. 3) Multimodal multiphoton imaging to explore the molecular mechanisms of demyelination. We have revealed a putative role of cytosolic phospholipase A2 in lysolecithin induced myelin vesiculation via real time CARS imaging of myelin degradation. We will pursue a systematic study of the molecular pathway in inflammation induced myelin damage. The proposed instrumentation activities are expected to push the capability of NLO microscopy to an unprecedented level. The methods being developed for in vivo imaging of CNS can be generally applied to other tissues such as tumor. Moreover, our development of advanced imaging technologies is strongly coupled to a compelling biomedical problem - demyelination. The proposed biomedical applications are expected to provide new knowledge about molecular mechanisms of demyelination. The new knowledge will help us to identify the key steps to be inhibited for alleviating myelin degradation in spinal cord injury and demyelinating diseases.